Production of Haploid Plant of 'Banpeiyu'pummelo [Citrus Maxima

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Production of Haploid Plant of ‘Banpeiyu’ Pummelo [Citrus maxima (Burm.) Merr.] by Pollination with Soft X-Ray-Irradiated Pollen

Masaki Yahata1**, Kiichi Yasuda1, Kohji Nagasawa2, Seiichi Harusaki2, Haruki Komatsu2 and Hisato Kunitake1*

1Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan 2School of Agriculture, Tokai University, Kumamoto 869-1404, Japan

To induce haploid plants in Citrus maxima (Burm.) Merr. ‘Banpeiyu’, we evaluated the effect of pollination with soft X-ray-irradiated pollen on fruit set and seed development, and carried out ovule culture. When ‘Banpeiyu’ pummelo pistils were pollinated with X-ray-irradiated pollen of ‘Fukuhara’ sweet orange [C. sinensis (L.) Osbeck], the exposure doses affected the fruit set. The number of seeds per fruit was also affected by the exposure dose, and tended to decrease as the dose increased; however, all developed seeds obtained from these crosses were diploid. In the ovule culture of ‘Banpeiyu’ pummelo, six embryoids shown haploidy were obtained in all treatments. One haploid plantlet with 9 chromosomes was regenerated from an embryoid in a culture of ovules established 40 days after pollination with 400 Gray (Gy)-irradiated pollen of ‘Tosa-buntan’ pummelo (C. maxima). This haploid was suggested to be derived from ‘Banpeiyu’ pummelo by random amplified polymorphic DNA (RAPD) and cleaved amplified polymorphic sequence (CAPS) analysis.

Key Words: chromosome observation, cleaved amplified polymorphic sequence (CAPS), flow cytometry, ovule culture, random amplified polymorphic DNA (RAPD).

Introduction hybridization (Germanà and Chiancone, 2001; Oiyama and Kobayashi, 1993; Toolapong et al., 1996); however, Haploids have significant potential in Citrus breeding, these alternative methods have not been effective, and because homozygous plants, which are very important so it was difficult to carry out genetic analysis and for genetic analysis and breeding, are easily obtained by breeding using haploids. doubling their chromosomes. Through the fusion of Recent biological studies of various plants suggest haploid protoplasts, new forms of interspecific and that parthenogenesis induced by the pollination of intergeneric hybrids can be obtained (Grosser et al., inactivated pollen with or without a pseudo-fertilized 2000). Furthermore, gene transfer or mutagenesis of ovule culture might represent an alternative technique haploids, followed by chromosome doubling, would for the production of haploid plants in plant species in enable recessive genes to be expressed. In Citrus, haploid which anther culture is inefficient. Sauton and Dumas seedlings were first obtained by the application of γ-rays (1987) first succeeded in producing haploid plants in in natsudaidai (C. natsudaidai Hayata) (Karasawa, several cultivars of melon (Cucumis melo L.) using the 1971). Esen and Soost (1971) described a haploid pseudo-fertilized ovule culture technique. They cultured embryo obtained from an immature seed of clementine immature ovules after fertilization with pollen irradiated mandarin (C. clementina hort. ex Tanaka) × ‘Pearl’ with 300 Gy of γ-rays from a cobalt 60 source. Katoh tangelo cross. Since then, haploids have been produced et al. (1993) also reported high-frequency haploid by anther culture (Germanà and Chiancone, 2003; production in melon by the same pseudo-fertilized ovule Germanà et al., 1994; Hidaka et al., 1979) and interploid culture (maximum 3.4% per examined ovule), although the frequency varied with the genotype. In fruit trees, haploid plants induced by irradiated pollen have been Received; June 24, 2009. Accepted; January 15, 2010. * Corresponding author (E-mail: [email protected]). reported for apple (Malus domestica Borkh.) (Zhang and ** Present address: Faculty of Agriculture, Shizuoka University, Lespinasse, 1991), pear (Pyrus communis L.) (Bouvier Shizuoka 422-8529, Japan. et al., 1993), and kiwifruit [Actinidia deliciosa (A. Chev.)

239 240 M. Yahata, K. Yasuda, K. Nagasawa, S. Harusaki, H. Komatsu and H. Kunitake

C. F. Liang & A. R. Ferguson] (Chalak and Legave, recorded. The seeds were extracted from each fruit and 1997; Pandey et al., 1990). classified into three groups, namely developed (normal In Citrus, on the other hand, there is only scant development), undeveloped (poor growth embryo), and information on parthenogenesis induced by inactivated other (empty and ovulelike) seeds. After all developed pollen (Froelicher et al., 2007; Ollitraut et al., 1996). seeds had been surface-dried and weighed and their seed Froelicher et al. (2007) produced five haploid plantlets coats had been removed, they were placed on double from three mandarin genotypes by pollinating with layers of moistened filter paper and kept at 25°C for pollen of Meyer lemon (C. meyeri Y. Tanaka) irradiated seven days. After germination, the seedlings were at 150 and 300 Gy of γ-rays from a cobalt 60 source. transplanted into vermiculite in boxes and transferred to In the present study, we describe in detail the effects a greenhouse. Undeveloped seeds were aseptically of pollination with soft X-ray-irradiated pollen on fruit cultured on Murashige and Skoog (MS) medium (1962) set and seed development, and demonstrate that containing 500 mg·L−1 malt extract, 30 g·L−1 sucrose, and pollination with irradiated pollen followed by ovule 2g·L−1 gellan gum without the seed coats being removed. culture in C. maxima was successful as a means of These cultures were kept at 25°C under continuous haploid production. illumination (38 μmol·m−2·s−1). Materials and Methods Ovule culture Plant materials Approximately 15 young fruits of ‘Banpeiyu’ ‘Banpeiyu’ pummelo, a monoembryonic diploid pummelo were collected 10, 20, and 40 days after cultivar, cultivated at the Fruit Research Institute, pollination with the 400 Gy-irradiated pollen of Kumamoto Prefectural Agricultural Research Center, ‘Fukuhara’ sweet orange and ‘Tosa-buntan’ pummelo. Matsubase, Kumamoto, Japan, was used as the seed ‘Banpeiyu’ pummelo fruits pollinated with non- parent. As pollen parents, ‘Fukuhara’ sweet orange and irradiated pollen of ‘Fukuhara’ sweet orange were used ‘Tosa-buntan’ pummelo, cultivated at the Citrus as controls. Ovules were excised aseptically from young Experimental Orchard, Faculty of Agriculture, Saga fruits with the aid of a dissecting microscope. The ovules University, were used. were cultured on Murashige and Tucker (MT) medium (1969) containing 500 mg·L−1 malt extract, 20 mg·L−1 Pollen irradiation adenine, 30 g·L−1 sucrose, and 2 g·L−1 gellan gum, and Collected pollen was placed on paraffin paper in a kept at 25°C under continuous illumination glass Petri dish (95 × 15 mm), which was transferred to (38 μmol·m−2·s−1). Callus, embryoid, and plant formation a soft X-ray unit (SV-100AW/I; Softex Co. Ltd., from these ovules was recorded 3 months after culture. Kanagawa, Japan) and irradiated with 0, 200, 400, 600, Plantlets obtained from the ovule culture were micro- 800, and 1000 Gy. grafted onto 20-day-old seedlings of trifoliate orange [Poncirus trifoliata (L.) Raf.]. Grafted plantlets were Pollen stainability and in vitro germination of irradiated directly transferred to sterilized soil in pots and grown pollen in a greenhouse, which was air-conditioned to maintain ‘Fukuhara’ sweet orange was used for testing pollen a temperature of about 20°C. stainability and in vitro germination of irradiated pollen. Pollen stainability was estimated with acetocarmine Ploidy level analysis crushing of nearly mature anthers. In vitro germination Flow cyotometry of pollen grains was performed on a glass slide covered Tissue segments were collected from the embryoids with a 2 mm layer of 1% (w/v) agar medium containing or developing leaves of a plantlet, chopped with a razor 10% sucrose. Five dehisced anthers, each from different blade in 2 mL buffer solution containing 1.0% (v/v) flowers, were rubbed on the agar medium, and the slides Triton X-100, 140 mM mercaptoethanol, 50 mM were then incubated for 10 h in a moistened chamber at Na2SO3, and 50 mM Tris-HCl at pH 7.5, and incubated 25°C in the dark. For each irradiation dose, pollen for 5 min according to the preparation method of Yahata stainability and in vitro germination rates were evaluated et al. (2005a). Crude samples were filtered through from three hundred grains with three repetitions. Miracloth (Merck KGaA, Darmstadt, Germany) and stained with 25 μg·L−1 propidium iodide (PI). The Effect of pollination with soft X-ray-irradiated pollen on relative fluorescence of total DNA was measured for fruit and seed development each nucleus with a Flow Cytometry System (EPICS Twenty-five flowers of ‘Banpeiyu’ pummelo were XL; Beckman Coulter, Fullerton, CA, USA) equipped hand-pollinated with pollen of ‘Fukuhara’ sweet orange with an argon laser (488 nm, 15 mW). irradiated at each exposure rate, and were immediately Chromosome observation covered with paraffin paper bags after emasculation. Young leaves (approximately 3–5 mm long) were Fruits were harvested at maturity between November excised from the plantlet, immersed in 2 mM 8- and early January, and fruit sets and weights were hydroxyquinoline for 6 h at 4°C, and fixed in a mixed J. Japan. Soc. Hort. Sci. 79 (3): 239–245. 2010. 241 solution of ethanol and acetic acid (3 : 1) for 12 h at 4°C. 11.8% in vitro germination (Table 1). There were no Enzymatic maceration and air drying were performed significant differences in stainability and in vitro according to the method of Fukui (1996) with some germination rates among the different doses of soft X- modifications. Young leaves were washed in distilled ray irradiation. It has been shown that irradiation of water to remove the fixative and then macerated in an pollen hardly affects stainability or in vitro germination enzyme mixture containing 2% (w/v) Cellulase Onozuka in apple (Zhang and Lespinasse, 1991), melon (Sauton RS (Yakult Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), and Dumas, 1987), kiwifruit (Musial and Przywara, 1% (w/v) Macerozyme R-200 (Yakult Pharmaceutical 1998) and cacao (Theobroma cacao L.) (Falque et al., Ind. Co. Ltd.), 0.3% Pectolyase (w/v) (Kyowa Chemical 1992). In Citrus, Froelicher et al. (2007) reported that Products Co. Ltd., Osaka, Japan), and 200 mM EDTA in vitro pollen germination of Meyer lemon was not at 37°C for 40 min. The macerated samples were rinsed affected at irradiation doses of 0–900 Gy γ-rays. On the with distilled water, and a fixative solution was added. other hand, De Lange and Vincent (1988) reported that The mixtures were transferred to glass slides. After the in vitro pollen germination of trifoliate orange, pummelo, slides had been air dried, the chromosomes were stained and sweet lime (C. aurantifolia (Cristm.) Swingle) with 2% Giemsa solution (Merck KGaA) in 1/30 decreased at irradiation doses above 500 Gy γ-rays. The phosphate buffer (pH 6.8) for 30 min, rinsed with response of pollen to irradiation might depend on the distilled water, air dried, and observed under an optical plant genotype and the type of irradiation applied. microscope. Effect of pollination with soft X-ray-irradiated pollen on Origin of haploid fruit and seed development Total DNA was extracted from young leaves of When pistils of ‘Banpeiyu’ pummelo were pollinated ‘Banpeiyu’ pummelo, ‘Tosa-buntan’ pummelo, and the with pollen of ‘Fukuhara’ sweet orange irradiated at haploid according to the method of Doyle and Doyle various doses of soft X-ray exposure, the fruit set and (1987). Total DNA was used for analyses of nuclear weight differed depending on the amount of radiation DNA by random amplified polymorphic DNA (RAPD) (Table 2), i.e., the percentage of fruit set and average and cleaved amplified polymorphic sequence (CAPS) fruit weight obtained from flowers pollinated with non- analysis. RAPD analysis of nuclear DNA was performed irradiated pollen were 37% and 1200 g, respectively, by a modified method of Williams et al. (1990). The whereas those from flowers pollinated with irradiated primers used were OPA20 in Operon random 10-mer pollen varied from 20 to 32% and 894 to 1115 g, primers (Operon Technologies, Inc., Huntsville, AL, USA). CAPS analysis was performed for the internal transcribed spacer (ITS) region in nuclear ribosomal Table 1. Effect of soft X-ray exposure dose on pollen stainability and in vitro RNA (rRNA). ITS1 and ITS4 were used as primers germination of ‘Fukuhara’ sweet orange. (Yasui et al., 1998). For each combination of samples Soft X-ray exposure Stainability in vitro germination and primers, PCR was carried out twice, and only stable dose (Gy) (%) (%) polymorphisms were analyzed. 0 82.5 10.2 200 87.6 11.4 Results and Discussion 400 78.5 8.2 Pollen stainability and in vitro germination 600 85.6 11.8 Non-irradiated pollen of ‘Fukuhara’ sweet orange had 800 82.5 10.7 a stainability rate of 82.5% and an in vitro germination 1000 84.5 10.0 rate of 10.2%, whereas pollen irradiated at doses ranging NSz NS

200–1000 Gy showed 78.5–87.6% stainability and 8.2– z Mean separation by Tukey’s multiple range test, P = 0.01.

Table 2. Fruits set, seed contents, and ploidy level of seedlings in the cross of ‘Banpeiyu’ pummelo with ‘Fukuhara’ sweet orange pollen irradiated by various doses of soft X-ray exposure. No. of X-ray No. of Av. fruit wt. No. of seeds per fruit No. of seedlings No. of seedlings exposure flowers fruits (g) obtained dose (Gy) pollinated set Developed Undeveloped X 2X 3X Unidentified 0 30 11 (36.7%) 1200.3az 381 24 Not observed Not observed 200 25 7 (28.0%) 1115.3a 25 75 22 0 21 0 1 400 25 8 (32.0%) 1081.3a 6 44 6 0 4 0 2 600 25 5 (20.0%) 893.6b 0 8 0 — — — — 800 25 6 (24.0%) 926.1ab 0 0 0 — — — — 1000 25 5 (20.0%) 1152.3a 0 0 0 — — — — z Mean separation by Tukey’s multiple range test, P = 0.01. 242 M. Yahata, K. Yasuda, K. Nagasawa, S. Harusaki, H. Komatsu and H. Kunitake respectively. The number of seeds per fruit also depended diploid, and undeveloped seeds did not germinate. In on the exposure dose, tending to decrease with each Citrus, Froelicher et al. (2007) also reported that the increase of exposure dose (Fig. 1). Although developed number of normal seeds decreased and the number of seeds were obtained from ‘Banpeiyu’ pummelo fruits small seeds increased when three mandarin cultivars, that underwent 200 and 400 Gy treatments, no developed ‘Ellendale’ tangor, ‘Fortune’ mandarin, and clementine seed was obtained from treatments over 600 Gy. All mandarin, were pollinated with Meyer lemon pollen seedlings obtained from developed seeds were diploid; irradiated at 150 and 300 Gy of γ-rays. In the meantime, no haploid seedlings were obtained in any treatments. they succeeded in the induction of five haploid plants Moreover, undeveloped in vitro cultured seeds did not from these mandarin cultivars by the culture of small germinate at all. seeds obtained from pollination by irradiated pollen at The effect of pollination with irradiated pollen on fruit 150 and 300 Gy. This tendency was observed at doses set, seed numbers, fruit and seed development has been ranging from 200 to 600 Gy in the present study, although investigated in many fruit trees, and the irradiation dose no haploid was obtained from undeveloped seeds. has been a factor (De Lange and Vincent, 1988; Presumably, the embryos of many undeveloped seeds Froelicher et al., 2007; Pandey et al., 1990). Furthermore, obtained in the present study may be considered to have the process of embryo development has been observed been haploid. In ‘Banpeiyu’ pummelo, it may be possible by histological analysis in various fruit trees (Musial to obtain haploid plants using pollen irradiated by soft and Przywara, 1998; Ogata et al., 2008; Peixe et al., X-ray at doses ranging from 200 to 600 Gy combined 2000; Sniezko and Visser, 1987; Zhang and Lespinasse, with ovule culture at an early stage of embryo 1991). It has been demonstrated that abnormal development. development of endosperm caused seed abortion in these observations. In the present study, with the increased Ovule culture irradiation dose, the number of developed seeds The induction of a haploid plant via ovule culture was decreased and undeveloped seeds increased, as in carried out because no haploid plants were recovered previous studies; however, the developed seeds were all from seeds following pollination with soft X-ray- irradiated pollen. After 3 months of ovule culture, callus formation from the integument was observed (Table 3). When the culture of ovules was initiated 10 days after pollination, the frequency of callus formation was very low, 0.5% for irradiated pollen of ‘Fukuhara’ sweet orange and 0.6% for that of ‘Tosa-Buntan’ pummelo, whereas callus formation in the culture of ovules initiated 40 days after pollination was 66.3% for ‘Fukuhara’ sweet orange and 61.1% for ‘Tosa-Buntan’ pummelo. Callus formation depended on the time of ovule culture, and it tended to increase with the progress in time for all treatments. On the other hand, the frequency of embryoid formation was generally very low; a total of eight Fig. 1. Seed development in ‘Banpeiyu’ pummelo crossed with embryoids was obtained from all treatments (Fig. 2A). ‘Fukuhara’ sweet orange pollen irradiated by various doses of The highest frequency (0.4%) was obtained when ovule soft X-ray exposure (Bar = 5cm). culture was initiated 40 days after pollination with

Table 3. Effect of pollen parents and duration after pollination on the haploid production of ‘Banpeiyu’ pummelo.

X-ray exposure Day after No. of ovules No. of ovules No. of embryoid Response Pollen parent z dose (Gy) pollination cultured forming callus formation Embryo Plantlet 10 708 7 (1.0%) 0 (0%) — — ‘Fukuhara’ 0 20 845 31 (3.7%) 0 (0%) — — 40 891 775 (87.0%) 1 (0.1%) 1 0 10 921 5 (0.5%) 3 (0.3%) 3 (2)y 0 ‘Fukuhara’ 400 20 917 18 (2.0%) 0 (0%) — — 40 841 558 (66.3%) 1 (0.1%) 1 (1) 0 10 879 5 (0.6%) 0 (0%) — — ‘Tosa-Buntan’ 400 20 833 20 (2.4%) 0 (0%) — — 40 947 579 (61.1%) 4 (0.4%) 3 (2) 1 (1) z Integument-derived callus. y Values in parentheses show the number of haploids. J. Japan. Soc. Hort. Sci. 79 (3): 239–245. 2010. 243

400 Gy-irradiated pollen of ‘Tosa-buntan’ pummelo. The those of ‘Banpeiyu’ pummelo, and no unique bands from difference in pollen parents did not significantly affect ‘Tosa-buntan’ pummelo were formed in the haploid the frequency of the callus and embryoid formation. (Figs. 4 and 5). These results indicate that this haploid When the embryoids were transferred to plant growth was of maternal origin. This haploid plantlet was micro- regulator-free MT medium, one plantlet was regenerated grafted onto trifoliate orange because it showed a very from an embryoid in an ovule culture initiated 40 days slow growth rate (Fig. 2D). After grafting, the haploid culture after pollination with 400 Gy-irradiated pollen grew vigorously (Fig. 6); moreover, five haploid of ‘Tosa-buntan’ pummelo (Fig. 2B). In other strains, embryoids died in culture. the embryoids showed enlarged cotyledonary tissue with Germanà (2006) demonstrated that the success of this the development of only an adventitious bud or root technique is dependent on the choice of the radiation (Fig. 2C). Flow cytometric analysis of one regenerated dose, the developmental stage of the embryos at the time plantlet and five embryoids revealed that they had of culture, the culture conditions, and the medium fluorescence intensity half that of ‘Banpeiyu’ pummelo requirements. In the present study, ovule culture of 40 (Fig. 3A). Chromosome observation of immature leaflets days after pollination with irradiated pollen gave the of the plantlet revealed that it was haploid with 9 highest frequency of haploid plants and embryoid chromosomes (Fig. 3B). Furthermore, RAPD and CAPS recovery. Yang (1968) reported that fertilized eggs began analysis confirmed DNA amplifications. The haploid to divide 40 to 50 days after pollination in natsudaidai was found to have almost the same banding patterns as and satsuma mandarin (C. unshiu Marcow.). Ovule

Fig. 2. Ovule culture of ‘Banpeiyu’ pummelo after pollination with 400 Gy-irradiated pollen of ‘Tosa-buntan’ pummelo. A: Embryoids, 3 months after culture (Bar = 1 cm). B: A plantlet developed from the embryoid (Bar = 1 cm). C: Abnormal embryoid (Bar = 1 cm). D: 3 weeks after in vitro grafting of the plantlet (Bar = 1cm).

Fig. 3. Flow cytometric analysis and chromosome observation of the plantlet developed from the embryoid of ‘Banpeiyu’ pummelo. A: Flow cytometric analysis. B: Chromosomes of young leaf cells (2n = X = 9, Bar = 10 μm). Z ‘Banpeiyu’ pummelo was used as a control. 244 M. Yahata, K. Yasuda, K. Nagasawa, S. Harusaki, H. Komatsu and H. Kunitake

Fig. 4. Random amplified polymorphic DNA (RAPD) analysis of ‘Banpeiyu’ pummelo, the haploid, and ‘Tosa-buntan’ pummelo. M: 100-bp ladder marker. B: ‘Banpeiyu’ pummelo. H: Haploid. T: ‘Tosa-buntan’ pummelo. Arrows indicate bands specific to Fig. 6. The haploid plant of ‘Banpeiyu’ pummelo, 5 years after each parent. grafting (Bar = 20 cm).

of haploid induction, such as selection of the seed and pollen parents, and the culture conditions in the ovule culture of C. maxima (Burm.) Merr. In Citrus and related genera, haploid production has been reported in natsudaidai, trifoliate orange, clementine mandarin, and ‘Lee’ (Germanà and Chiancone, 2001, 2003; Germanà et al., 1994; Hidaka et al., 1979; Karasawa, 1971; Oiyama and Kobayashi, 1993). These haploids were very weak, and their growth was slower than that of diploid plants. On the other hand, the haploid of ‘Banpeiyu’ pummelo produced by Toolapong et al. (1996) showed vigorous growth and flowered for the first time seven years after germination. This haploid plant was confirmed to produce fertile pollen (Yahata et al., 2005a), and several diploid progenies were obtained following the pollination of four diploid plants with pollen from this haploid (Yahata et al., 2005b). The haploid of ‘Banpeiyu’ pummelo obtained in the present study is expected to produce flowers because it has shown vigorous growth similar to the haploid of ‘Banpeiyu’ pummelo produced by Toolapong et al. Fig. 5. Restriction pattern of HaeIII-digested ribosomal RNA (rRNA) (1996). internal transcribed spacer (ITS) of nuclear genomes. M: 100- In conclusion, we obtained a haploid plant and some bp ladder marker. B: ‘Banpeiyu’ pummelo. H: Haploid. T: ‘Tosa- buntan’ pummelo. Arrows indicate bands specific to each parent. haploid embryoids by means of ovule culture in ‘Banpeiyu’ pummelo. The haploid plant obtained in the present study will be valuable for genetic analysis and culture at approximately 40 days after pollination seems possibly for planned breeding, and useful material for to be appropriate for the haploid induction of Citrus. triploid seedless breeding by somatic hybridization of Further study is needed to clarify the optimum conditions haploid and diploid protoplasts. J. Japan. Soc. Hort. Sci. 79 (3): 239–245. 2010. 245

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